All aerobic organisms produce reactive oxygen species (ROS) including superoxide anion. Superoxide can arise inside the cell as a byproduct of metabolism and is also generated extracellularly to combat pathogens and act in signaling. To manage this important ROS, aerobic organisms have evolved with various forms of metal containing superoxide dismutases (SOD). The most prominent SODs in eukaryotes are the Cu/Zn isoforms including intracellular SOD1 and extracellular EC-SOD. Up until 2014, these were thought to be the only Cu SODs in eukaryotes, but we recently uncovered a new family that deviates from SOD1 and EC-SOD in terms of tertiary and quaternary structure as well as metal ion requirement. The prototype is extracellular SOD5 from the human fungal pathogen Candida albicans. Unlike SOD1 and EC-SOD, SOD5 is monomeric, lacks Zn and the electrostatic loop for substrate guidance, and yet reacts with superoxide at rates that match Cu/Zn SODs. SOD5-like SODs occur throughout fungi and we have identified related proteins in animals. In all cases, these are cell surface extracellular proteins predicted to react with extracellular ROS. In this research proposal, we aim to unravel the metallobiochemistry, structural and cellular biology of this new family of cuproproteins in eukaryotes. Aim 1: To define SOD5's capacity for functioning at the host-pathogen interface: We hypothesize that the SOD5 metalloenzyme is ideally suited to function under the harsh environment of the host-pathogen interface. This shall be addressed using recombinant proteins to examine SOD5's metal binding properties, susceptibility to proteases and reactivity with oxidants such as H2O2. Findings will be compared to mammalian EC-SOD and extrapolated to a C. albicans macrophage infection system where SOD5 and EC-SOD naturally co-exist. Aim 2: To understand the origins of C. albicans ROS as substrates for extracellular SOD5: SOD5 is well known to combat the superoxide attack of the animal host and we recently identified another substrate for SOD5, namely a burst of superoxide produced by the yeast itself during differentiation. We will use biochemical and genetic approaches to identify the putative fungal oxidoreductase(s) responsible for this yeast ROS and explore how the opposing forces of fungal ROS and fungal SOD5 impact C. albicans invasion of macrophages. Aim 3: To understand the function of CSRP molecules in animals: We identified SOD5-like sequences in numerous animal genomes, but rather than single domain enzymes, the sequences occur as 4x tandem repeats in large extracellular proteins we call CSRP (Cu-SOD repeat proteins). Could this be a new form of SOD metalloenzyme that functions as a repeating unit? Using recombinant CSRP from Zebrafish and Anophoes gambiae we will examine CSRP metal binding properties and possible SOD activity, and work towards defining their structure and function in animal cells. The extended family of SOD5-like proteins have certainly been diversified in nature to function as either single unit SOD enzymes or as repeated metalloprotein domains for redox and/or metallobiology.